Method for producing hydroxy-l-pipecolic acid

ABSTRACT

A novel method of producing high-purity hydroxy-L-pipecolic acids in an efficient and inexpensive manner while suppressing the production of hydroxy-L-proline is provided. The method includes allowing an L-pipecolic acid hydroxylase, a microorganism or cell having the ability to produce the enzyme, a processed product of the microorganism or cell, and/or a culture liquid comprising the enzyme and obtained by culturing the microorganism or cell, to act on L-pipecolic acid as a substrate in the presence of 2-oxoglutaric acid and ferrous ion, wherein the L-pipecolic acid hydroxylase has the properties:
         (1) the enzyme can act on L-pipecolic acid in the presence of 2-oxoglutaric acid and ferrous ion to add a hydroxy group to the carbon atom at positions 3, 4, and/or 5 of L-pipecolic acid; and   (2) the enzyme has a catalytic efficiency (kcat/Km) with L-proline that is equal to or less than 7 times the catalytic efficiency (kcat/Km) with L-pipecolic acid.

TECHNICAL FIELD

The present invention relates to a method of biologically producinghydroxy-L-pipecolic acids, particularly to a method of producinghydroxy-L-pipecolic acids from L-pipecolic acid based on the use ofenzymes that have the ability to produce the hydroxy-L-pipecolic acids.

BACKGROUND ART

Hydroxy-L-pipecolic acids are compounds useful as intermediates toproduce pharmaceuticals, and the like. It is known that, for example,(4S)-hydroxy-L-pipecolic acid and (4R)-hydroxy-L-pipecolic acid can beused as precursors of a Rho kinase inhibitor (Patent Document 1) and theHIV protease inhibitor palinavir (Non-Patent Document 1), respectively,while (5S)-hydroxy-L-pipecolic acid and (5R)-hydroxy-L-pipecolic acidcan be used as precursors of antimicrobials (Patent Document 2).

It has been known that hydroxy-L-pipecolic acids can be produced fromL-pipecolic acid by biological approaches. Examples of proteins havingthe ability to convert L-pipecolic acid to (5S)-hydroxy-L-pipecolic acidreportedly include the BAB52605 protein derived from the root nodulebacterium Mesorhizobium loti strain MAFF303099 isolated from Lotusjaponicus, the CAC47686 protein derived from the root nodule bacteriumSinorhizobium meliloti strain 1021 isolated from Medicago sativa(hereinafter sometimes referred to as “SmPH”), and a protein encoded ina polynucleotide (cistronic gene), the expression of which is initiated48 nucleotides (corresponding to 16 amino acids) upstream of thetranslation start site described in the annotation of the EFV12517protein derived from the Segniliparus rugosus strain ATCC BAA-974(hereinafter sometimes referred to as “SruPH”), and the like (PatentDocument 3). These hydroxylases can be used to convert L-pipecolic acidto hydroxy-L-pipecolic acids.

However, any of these hydroxylases also has the ability to producehydroxy-L-proline through the hydroxylation of L-proline.

An L-pipecolic acid biologically produced by using an enzyme, microbialbodies, or the like may be contaminated with a trace amount ofL-proline, for example, as a carry-over component from a medium used forthe preparation of the enzyme or the microbial bodies.

Moreover, in cases where hydroxy-L-pipecolic acids are produced frompure L-pipecolic acid as a base material by using the hydroxylases, thehydroxylases to be used may be contaminated with L-proline.

Furthermore, even if neither L-pipecolic acid as a base material nor thehydroxylases are contaminated with L-proline, in cases where thehydroxylation reaction is achieved by culturing microbial bodies capableof expressing the hydroxylases, L-proline may potentially be produced asa by-product by those microbial bodies during the course of theirgrowth.

Since the physicochemical properties of L-proline are close to those ofL-pipecolic acid, it is difficult to obtain pure L-pipecolic acid withcomplete removal of L-proline.

Moreover, when L-pipecolic acid contaminated with L-proline is used as abase material along with any of the aforementioned enzymes in theconversion of L-pipecolic acid to a hydroxy-L-pipecolic acid, L-prolineis converted to hydroxy-L-proline and, therefore, the resulting productwill be a mixture of the hydroxy-L-pipecolic acid and hydroxy-L-proline,which makes it difficult to obtain the hydroxy-L-pipecolic acid of highpurity. Also, since the physicochemical properties of hydroxy-L-prolineare close to those of hydroxy-L-pipecolic acids, it is difficult toseparate them from each other.

Accordingly, disadvantageously, a heavy burden in purification processand a huge expense of time and money have been required for thepreparation of high-purity hydroxy-L-pipecolic acids with removal ofhydroxy-L-proline because multiply repeated purification and use ofexpensive purification measures are required.

PRIOR ART DOCUMENTS Patent Documents

-   Patent Document 1: JP-T-2010-514720-   Patent Document 2: JP-T-2004-505088-   Patent Document 3: WO2013/187438

Non-Patent Document

-   Non-Patent Document 1: Gillard et al., The Journal of Organic    Chemistry, 1996, Vol. 61, pp. 2226

SUMMARY OF THE INVENTION Technical Problem

As described above, methods of biologically producinghydroxy-L-pipecolic acids from L-pipecolic acid are known; however, anyof those conventional methods is unsatisfactory as a production methodfor hydroxy-L-pipecolic acids of high purity, which are used asintermediates of pharmaceuticals, and the like, because not onlyhydroxy-L-pipecolic acids but also hydroxy-L-proline are produced bythose methods. Accordingly, there is a demand for a more efficientproduction method.

Namely, an object of the present invention is to provide a novel methodof producing high-purity hydroxy-L-pipecolic acids in an efficient andinexpensive manner while suppressing the production ofhydroxy-L-proline.

Solution to Problem

The inventors intensively studied to solve the above-described problemand consequently found that particular pipecolic acid hydroxylases havethe ability to convert L-pipecolic acid to hydroxy-L-pipecolic acids(L-pipecolic acid hydroxylation activity) in a 2-oxoglutarate-dependentfashion and have almost no activity to convert L-proline tohydroxy-L-proline (L-proline hydroxylation activity), and that varioushydroxy-L-pipecolic acids with high optical purity can be obtained in ahighly efficient manner by allowing those particular enzymes to act onL-pipecolic acid. The present invention was achieved based on thesefindings.

Now, the spirit of the present invention will be described below.

[1] A method of producing a hydroxy-L-pipecolic acid, the methodcomprising:

allowing an L-pipecolic acid hydroxylase, a microorganism or cell havingthe ability to produce the enzyme, a processed product of themicroorganism or cell, and/or a culture liquid comprising the enzyme andobtained by culturing the microorganism or cell, to act on L-pipecolicacid as a substrate in the presence of 2-oxoglutaric acid and ferrousion, wherein the L-pipecolic acid hydroxylase has the properties (1) and(2) below:

(1) the enzyme can act on L-pipecolic acid in the presence of2-oxoglutaric acid and ferrous ion to add a hydroxy group to the carbonatom at positions 3, 4, and/or 5 of L-pipecolic acid; and

(2) the enzyme has a catalytic efficiency (kcat/Km) with L-proline thatis equal to or less than 7 times the catalytic efficiency (kcat/Km) withL-pipecolic acid.

[2] The method of producing a hydroxy-L-pipecolic acid according to [1],further comprising:

allowing L-lysine and/or DL-lysine to react with

-   -   (i-1) one or more enzymes selected from the group consisting of        an L-amino acid oxidase, an L-amino acid dehydrogenase and an        L-amino acid aminotransferase, or    -   (i-2) an amino acid racemase and one or more enzymes selected        from the group consisting of a D-amino acid oxidase, a D-amino        acid dehydrogenase and a D-amino acid aminotransferase,        for the production of 3,4,5,6-tetrahydropyridine-2-carboxylic        acid; and subsequently

allowing an N-methyl-L-amino acid dehydrogenase to act on the3,4,5,6-tetrahydropyridine-2-carboxylic acid for the production of theL-pipecolic acid as the substrate.

[3] The method of producing a hydroxy-L-pipecolic acid according to [1],further comprising:

allowing L-lysine to react with one or more enzymes selected from thegroup consisting of an L-lysine 6-oxidase, an L-lysine 6-dehydrogenaseand an L-lysine 6-aminotransferase, for the production of2,3,4,5-tetrahydropyridine-2-carboxylic acid; and subsequently

allowing a pyrroline-5-carboxylate reductase to act on the2,3,4,5-tetrahydropyridine-2-carboxylic acid for the production of theL-pipecolic acid as the substrate.

[4] The method of producing a hydroxy-L-pipecolic acid according to [1],further comprising allowing a lysine cyclodeaminase to act on L-lysinefor the production of the L-pipecolic acid as the substrate.

[5] The method of producing a hydroxy-L-pipecolic acid according to anyof [1] to [4], wherein the content of L-proline in the L-pipecolic acidas the substrate is not more than 10% (w/w).

[6] The method of producing a hydroxy-L-pipecolic acid according to anyof [1] to [5], wherein the L-pipecolic acid hydroxylase further has theproperty (3) below:

(3) the microorganism or cell having the ability to produce theL-pipecolic acid hydroxylase, or the processed product of themicroorganism or cell has a hydroxy-L-proline-producing activity that isnot more than 55%, where a ratio of 100% corresponds to thehydroxy-L-pipecolic acid-producing activity of the same microorganism orcell, or of the same processed product.

[7] The method of producing a hydroxy-L-pipecolic acid according to anyof [1] to 161, wherein the L-pipecolic acid hydroxylase comprises theprotein (A), (B), or (C) below:

(A) a protein having an amino acid sequence represented by SEQ ID NO: 2,12, 14, 16, 18, or 20;

(B) a protein having the same amino acid sequence as the amino acidsequence represented by SEQ ID NO: 2, 12, 14, 16, 18, or 20 except thatone or several amino acids are deleted, substituted, inserted, and/oradded, which protein has the aforementioned properties (1) and (2);

(C) a protein having an amino acid sequence with an identity of not lessthan 80% to the amino acid sequence represented by SEQ ID NO: 2, 12, 14,16, 18, or 20, which protein has the aforementioned properties (1) and(2).

[8] The method of producing a hydroxy-L-pipecolic acid according to anyof [1] to [7], wherein the L-pipecolic acid hydroxylase, or themicroorganism or cell having the ability to produce the enzyme is amicroorganism or cell transformed with DNA encoding the L-pipecolic acidhydroxylase.[9] The method of producing a hydroxy-L-pipecolic acid according to 181,wherein the DNA encoding the L-pipecolic acid hydroxylase comprises theDNA (D), (E), or (F) below:

(D) DNA having a nucleotide sequence represented by SEQ ID NO: 1, 11,13, 15, 17, or 19;

(E) DNA comprising the same nucleotide sequence as the nucleotidesequence represented by SEQ ID NO: 1, 11, 13, 15, 17, or 19 except thatone or several bases are deleted, substituted, inserted, and/or added,which DNA encodes a protein having the aforementioned properties (1) and(2);

(F) DNA comprising a nucleotide sequence which hybridizes with acomplementary strand of the nucleotide sequence represented by SEQ IDNO: 1, 11, 13, 15, 17, or 19 under stringent conditions, which DNAencodes a protein having the aforementioned properties (1) and (2).

Advantageous Effects of the Invention

According to the present invention, high-purity hydroxy-L-pipecolicacids with high optical purity can be produced in a highly efficientmanner. In particular, according to the present invention, high-purityhydroxy-L-pipecolic acids can be produced even in cases where theL-pipecolic acid as the substrate is contaminated with L-proline.

Furthermore, the present invention is preferable from an industrialviewpoint because the production of hydroxy-L-proline can be reducedand, thus, high-purity hydroxy-L-pipecolic acids available asintermediates for pharmaceuticals, and the like can be produced at lowcost and at less expense.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents graphs obtained in Example 3 by plotting thereciprocals of the respective concentrations of L-pipecolic acid(mmol/L) on the x-axis and the reciprocals of the hydroxy-L-pipecolicacid-producing activities per milligram of each hydroxylase (U/mg) atthe respective concentrations of L-pipecolic acid on the y-axis.

FIG. 2 indicates the reactivity with TL-proline and L-pipecolic acid inL-pipecolic acid 5-hydroxylases compared in Example 4.

FIG. 3 shows the temporal change in amount of accumulated(5S)-hydroxy-L-pipecolic acid in Example 5.

FIG. 4 shows the temporal change in amount of accumulated(4S)-hydroxy-L-pipecolic acid in Example 5.

DESCRIPTION OF THE EMBODIMENTS

Now, the present invention will be described below in detail.

In the present specification, the “L-pipecolic acid hydroxylationactivity” means the ability to add a hydroxy group to the carbon atom atpositions 3, 4, and/or 5 of L-pipecolic acid.

Whether or not the “L-pipecolic acid hydroxylation activity” is retainedin a measurement subject can be confirmed, for example, by allowing anenzyme, which is a measurement subject, to act on L-pipecolic acid in areaction system containing L-pipecolic acid as a substrate and furthercontaining 2-oxoglutaric acid and ferrous ion, and then measuring theamount of resulting hydroxy-L-pipecolic acid.

The term “enzyme” in the present specification includes a purifiedenzyme (including a partially purified enzyme), and an immobilizedenzyme on a carrier prepared by using a conventional immobilizationtechnique, including, for example, an enzyme immobilized on a carriersuch as polyacrylamide and carrageenan gels, and the like.

In the present specification, the term “expression vector” refers to agenetic element that is used for the introduction of a polynucleotideencoding a protein with a desired function into a host organism to berecombined, followed by the replication of the polynucleotide and theexpression of the protein with the desired function in the hostorganism. Examples of the expression vector include, but are not limitedto, plasmid, virus, phage, cosmid, and the like, and a preferableexample is plasmid.

In the present specification, “transformant” means a microorganism orcell into which a gene of interest has been introduced using anexpression vector, such as those as described above, to allow themicroorganism or cell to exhibit a desired phenotype associated with anencoded protein with a desired function.

The method of producing a hydroxy-L-pipecolic acid according to thepresent invention is characterized by allowing an L-pipecolic acidhydroxylase, a microorganism or cell having the ability to produce theenzyme, a processed product of the microorganism or cell, and/or aculture liquid comprising the enzyme and obtained by culturing themicroorganism or cell, to act on the L-pipecolic acid as the substratein the presence of 2-oxoglutaric acid and ferrous ion, wherein theL-pipecolic acid hydroxylase has the properties (1) and (2) below:

(1) the enzyme can act on L-pipecolic acid in the presence of2-oxoglutaric acid and ferrous ion to add a hydroxy group to the carbonatom at positions 3, 4, and/or 5 of L-pipecolic acid; and

(2) the enzyme has a catalytic efficiency (kcat/Km) with L-proline thatis equal to, or less than 7 times the catalytic efficiency (kcat/Km)with L-pipecolic acid.

The L-pipecolic acid hydroxylase used in the present invention(hereinafter sometimes referred to as “L-pipecolic acid hydroxylase ofthe present invention”) is an enzyme capable of acting on L-pipecolicacid in the presence of 2-oxoglutaric acid and ferrous ion to add ahydroxy group to the carbon atom at positions 3, 4, and/or 5 ofL-pipecolic acid.

The amount of 2-oxoglutaric acid is not particularly limited as long asit is not an amount so as to inhibit the reaction, but it is normally anamount equimolar to or greater than the amount of L-pipecolic acid inthe reaction system and is preferably an amount ranging from 1 to 1.2times the number of moles of L-pipecolic acid in the reaction system.

The amount of ferrous ion is not particularly limited as long as it isnot an amount so as to inhibit the reaction, but it normally from 0.0001to 0.5 mol, preferably from 0.001 to 0.1 mol, per mol of L-pipecolicacid in the reaction system.

The addition of, for example, ferrous sulfate, ferrous chloride, ferrouscitrate, and the like allows ferrous ions to exist in the reactionsystem.

The L-pipecolic acid hydroxylase of the present invention preferablyincludes a protein having an amino acid sequence represented by SEQ IDNO: 2, 12, 14, 16, 18, or 20.

Moreover, the L-pipecolic acid hydroxylase of the present invention mayinclude a protein having an amino acid sequence homologous to the aminoacid sequence represented by SEQ ID NO: 2, 12, 14, 16, 18, or 20, whichis a protein having the L-pipecolic acid hydroxylation activity and theproperties (1) and (2) as described above.

Such a protein having an amino acid sequence homologous to the aminoacid sequence represented by SEQ TD NO: 2, 12, 14, 16, 18, or 20includes a protein having the same amino acid sequence as the amino acidsequence represented by SEQ ID NO: 2, 12, 14, 16, 18, or 20 except thatone or several amino acids are deleted, substituted, inserted, and/oradded. In the case of substitution, insertion, or addition, conservativemutations resulting from conservative substitution, insertion, oraddition of one or several amino acids are preferable.

The phrase “one or several amino acids” herein means usually 1 to 100,preferably 1 to 50, more preferably 1 to 20, still more preferably 1 to10, particularly preferably 1 to 5, amino acids.

Moreover, the protein having an amino acid sequence homologous to theamino acid sequence represented by SEQ ID NO: 2, 12, 14, 16, 18, or 20includes a protein having an amino acid sequence with an identity of notless than 70% to the amino acid sequence represented by SEQ ID NO: 2,12, 14, 16, 18, or 20 in its full-length form. The protein includes aprotein having an amino acid sequence with an identity of preferably notless than 80%, more preferably not less than 90%, and still morepreferably not less than 95% to the above-described amino acid sequencein its full-length form.

In the present invention, the term “sequence identity” means, innucleotide sequences or amino acid sequences, the percentage ofidentical nucleotides or amino acids shared between two sequences, whichpercentage is determined by aligning those two sequences in an optimalpairwise alignment. That is, the identity can be calculated with theformula below and can be calculated by using a commercially availablealgorithm:

Identity−(the number of matches at each position/the total number ofpositions)×100.

Moreover, such algorithms are incorporated in NBLAST and XBLAST programsas described in Altschul et al., J. Mol. Biol. 215 (1990) pp. 403-410.More particularly, the identity search and the identity analysis can beperformed on nucleotide sequences or amino acid sequences by means ofalgorithms or programs well known to those skilled in the art (forexample, BLASTN, BLASTP, BLASTX, ClustalW). Parameters for use with aprogram can be appropriately set by those skilled in the art and defaultparameters for each program may also be used. Specific procedures inthose analysis methods are well known to those skilled in the art.

The amino acid sequences represented by SEQ ID NO: 2, 12, 14, 16, and 18are based on the genome information of Micromonospora chokoriensis, theColletotrichum gloeosporioides strain Nara qc5, the Penicilliumchrysogenum strain Wisconsin 54-1255, the Gibberella zeae strain PH-1,and a Kordia jejudonensis strain, respectively.

Among those L-pipecolic acid hydroxylases of the present invention, theprotein having the amino acid sequence represented by SEQ ID NO: 2, 18,or 20, and the protein having an amino acid sequence homologous to theamino acid sequence represented by SEQ ID NO: 2, 18, or 20 and havingthe L-pipecolic acid hydroxylation activity selectively hydroxylates thecarbon atom at position 5 of L-pipecolic acid and therefore they canproduce (5S)-hydroxy-L-pipecolic acid with high efficiency.

Among those L-pipecolic acid hydroxylases of the present invention, theprotein having the amino acid sequence represented by SEQ ID NO: 12, 14,or 16, and the protein having an amino acid sequence homologous to theamino acid sequence represented by SEQ ID NO: 12, 14, or 16 and havingthe L-pipecolic acid hydroxylation activity selectively hydroxylates thecarbon atom at position 4 of L-pipecolic acid and therefore they canproduce (4S)-hydroxy-L-pipecolic acid with high efficiency.

In the present invention, one, two or more types of L-pipecolic acidhydroxylases can be used.

The L-pipecolic acid hydroxylase of the present invention can beobtained by purification from Micromonospora chokoriensis, theColletotrichum gloeosporioides strain Nara qc5, the Penicilliumchrysogenum strain Wisconsin 54-1255, the Gibberella zeae strain PH-1,the Kordia jejudonensis strain, or the like.

Also, the L-pipecolic acid hydroxylase of the present invention can beobtained by cloning a DNA encoding the L-pipecolic acid hydroxylase ofthe present invention by means of known methods including PCR(polymerase chain reaction), hybridization, and the like, and thenallowing the L-pipecolic acid hydroxylase to be expressed in anappropriate host.

The DNAs encoding L-pipecolic acid hydroxylases comprising the proteinshaving the amino acid sequences represented by SEQ ID NO: 2, 12, 14, 16,18, and 20 respectively correspond to, for example, DNAs comprising thenucleotide sequences represented by SEQ ID NO: 1, 11, 13, 15, 17, and19. Moreover, the DNA may be a DNA homologous to the above DNAcomprising the nucleotide sequence represented by SEQ ID NO: 1, 11, 13,15, 17, or 19, which encodes a protein having the L-pipecolic acidhydroxylation activity and the properties (1) and (2) as describedabove.

Such a DNA homologous to the above DNA comprising the nucleotidesequence represented by SEQ ID NO: 1, 11, 13, 15, 17, or 19 includes aDNA comprising the same nucleotide sequence as the nucleotide sequencerepresented by SEQ ID NO: 1, 11, 13, 15, 17, or 19 except that one orseveral bases are deleted, substituted, inserted, and/or added. In thecase of substitution, insertion, or addition, conservative mutationsresulting from conservative substitution, insertion, or addition of oneor several bases are preferable.

The phrase “one or several bases” herein means usually 1 to 300,preferably 1 to 150, more preferably 1 to 60, still more preferably 1 to30, more preferably 1 to 15, particularly preferably 1 to 10, bases.

The nucleotide sequences represented by SEQ ID NO: 1, 11, 13, 15, and 17respectively correspond to the nucleotide sequences of genes originatedfrom Micromonospora chokoriensis, the Colletotrichum gloeosporioidesstrain Nara qc5, the Penicillium chrysogenum strain Wisconsin 54-1255,the Gibberella zeae strain PH-1, and the Kordia jejudonensis strain,which nucleotide sequences are codon-optimized for E. coli expressionand encode the amino acid sequences represented by SEQ ID NO: 2, 12, 14,16, and 18. Such a DNA with codon-optimization according to the host fortransformation is, of course, included in examples of the DNA encodingthe L-pipecolic acid hydroxylase of the present invention.

Moreover, the DNA homologous to the above DNA comprising the nucleotidesequence represented by SEQ ID NO: 1, 11, 13, 15, 17, or 19 may be a DNAcomprising a nucleotide sequence which hybridizes with the complementarystrand of the nucleotide sequence represented by SEQ ID NO: 1, 11, 13,15, 17, or 19 under stringent conditions.

The phrase “nucleotide sequence which hybridizes under stringentconditions” herein means the nucleotide sequence of a DNA that isobtained by using a DNA probe and using colony hybridization, plaquehybridization, Southern blot hybridization, or the like under stringentconditions.

Examples of the stringent conditions include, in cases of colonyhybridization or plaque hybridization, conditions where filters withimmobilized DNA from colonies or plaques, or with immobilized DNAfragments thereof are used to perform hybridization at 65° C. in thepresence of 0.7 mol/L to 1 mol/L aqueous solution of sodium chloride,and the filters are subsequently washed under a temperature condition of65° C. by using 0.1×SSC solution (the composition of 1×SSC: 150 mmol/Laqueous solution of sodium chloride, 15 mmol/L aqueous solution ofsodium citrate). Such hybridization can be performed in accordance withthe methods described in Molecular Cloning: A laboratory Manual, 2^(nd)Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989, andthe like.

Those skilled in the art will be able to introduce a desired mutationinto the DNA comprising the nucleotide sequence represented by SEQ IDNO: 1, 11, 13, 15, 17, or 19 by deletion, substitution, insertion,and/or addition of one or more bases by means of site-directedmutagenesis (Nucleic Acids Res. 10, pp. 6487 (1982); Methods in Enzymol.100, pp. 448 (1983); Molecular Cloning; PCR: A Practical Approach, IRLPress, pp. 200 (1991)) and the like to provide a mutant DNA of the DNAcomprising the nucleotide sequence represented by SEQ ID NO: 1, 11, 13,15, 17, or 19.

Moreover, the amino acid information of the L-pipecolic acid hydroxylaseof the present invention or the nucleotide sequence information of a DNAencoding the same may be obtained by a homology search of the entire orpart of the amino acid sequence represented by SEQ ID NO: 2, 12, 14, 16,18, or 20, or the entire or part of the nucleotide sequence representedby SEQ ID NO: 1, 11, 13, 15, 17, or 19, against databases such as DNADatabank of JAPAN (DDBJ).

Moreover, the L-pipecolic acid hydroxylase of the present invention hasa catalytic efficiency (kcat/Km) with L-proline that is equal to, orless than 7 times, preferably equal to, or less than 2 times, morepreferably equal to, or less than 1 time, still more preferably equalto, or less than 0.5 times, and particularly preferably equal to, orless than 0.1 times the catalytic efficiency (kcat/Km) with L-pipecolicacid. An L-pipecolic acid hydroxylase exhibiting virtually nohydroxy-L-proline-producing activity is most preferable. The phrase“virtually no hydroxy-L-proline-producing activity” in the presentinvention does not mean not producing even one molecule ofhydroxy-L-proline but means producing an undetectable amount of, or adetectable but trace amount of hydroxy-L-proline, which is almost anegligible amount based on the common knowledge of those skilled in theart.

The catalytic efficiency (kcat/Km) in the present invention can bedetermined, for example, by a method using the Michaelis-Menten kineticequation to calculate the kcat/Km value. Herein, the term “kcat” refersto the catalytic efficiency per molecule of an enzyme, while the term“Km” is an index representing the affinity of the enzyme with itssubstrate.

A specific method to calculate Km and kcat is as described below. First,the hydroxy-L-pipecolic acid-producing activity per milligram of eachhydroxylase (U/mg) at each concentration of L-pipecolic acid is measuredin a particular range of substrate concentration. One unit (U) hereinrepresents the ability to produce 1 μmol of a hydroxy-L-pipecolic acidin the period of 1 minute. Subsequently, the reciprocals of therespective concentrations of L-pipecolic acid (mmol/L) and thereciprocals of the hydroxy-L-pipecolic acid-producing activities permilligram of the hydroxylase (U/mg) at the respective concentrations ofL-pipecolic acid are plotted on the x-axis and the y-axis, respectively.It is known that, in those linear plots, the reciprocal of eachintercept with the x-axis, multiplied with −1, represents a km value(mmol/L) and the reciprocal of each intercept with the y-axis representsa Vmax value (U/mg), the maximum rate of reaction. The catalytic abilityper molecule of the enzyme (kcat) can be calculated from the Vmax andthe molecular weight of the enzyme. The thus-obtained kcat and km valuescan be used to calculate the catalytic efficiency (kcat/Km), which is anindex representing the catalytic ability of the enzyme.

As an enzyme to be used for the calculation of the catalytic efficiency,a crudely or finely purified material containing an enzyme fractionextracted from the microorganism or cell having the ability to producethe enzyme, which are disrupted by treatment with an organic solvent, asurfactant, and the like, and/or by physical or enzymatic treatment, canbe used. The level of the enzymatic activity and the reaction time canbe set as desired to the extent that the enzymatic activity is increasedproportionally to the amount of the reaction time.

Details of the Michaelis-Menten equation and the like are described in,for example, “Biochemical Research Methods, Vol. 21: Guide toExperiments on Enzymatic Reaction Kinetics, Gakkai Shuppan Center, Ltd.”and the like.

Furthermore, the L-pipecolic acid hydroxylase activity in the presentinvention may also be simply evaluated by the following procedures.

The microorganism or cell having the ability to produce the L-pipecolicacid hydroxylase of the present invention, or the processed product ofthe microorganism or cell is allowed to react with L-pipecolic acid orL-proline and then the hydroxy-L-pipecolic acid-producing activity (U/g)and the hydroxy-L-proline-producing activity (U/g) per amount ofinputted cells (unit: gram (g) or turbidity) or per mass of totalproteins (unit: g) are measured in particular substrate concentrations.Then, the activity to produce hydroxy-L-proline from L-proline at aparticular substrate concentration (relative value) is calculated, wherea ratio of 100% corresponds to the activity to produce ahydroxy-L-pipecolic acid from L-pipecolic acid at the same substrateconcentration.

As for the L-pipecolic acid hydroxylase of the present invention, theactivity to produce hydroxy-L-proline in the microorganism or cellhaving the ability to produce the L-pipecolic acid hydroxylase, or inthe processed product of the microorganism or cell is normally not morethan 55%, preferably not more than 50%, more preferably not more than35%, particularly preferably not more than 20%, and most preferably 0%relative, where a ratio of 100% corresponds to the activity to produce ahydroxy-L-pipecolic acid in the microorganism or cell having the abilityto produce the enzyme, or in the processed product of the microorganismor cell.

Because the L-pipecolic acid hydroxylase used in the present invention(hereinafter sometimes referred to as “L-pipecolic acid hydroxylase ofthe present invention”) can hydroxylate L-pipecolic acid with highregiospecificity and high stereospecificity, hydroxy-L-pipecolic acidswith high optical purity can be obtained with high efficiency.

In the method of producing a hydroxy-L-pipecolic acid according to thepresent invention, the L-pipecolic acid hydroxylase of the presentinvention may be directly used for the reaction, or a microorganism orcell having the ability to produce the enzyme, a processed product ofthe microorganism or cell, and/or a culture liquid comprising the enzymeand obtained by culturing the microorganism or cell may be used for thereaction.

The microorganism or cell having the ability to produce the L-pipecolicacid hydroxylase of the present invention may be a microorganism or cellinherently having the above production ability, or a microorganism orcell on which the above production ability has been conferred bybreeding. Moreover, the microorganism or cell having the ability toproduce the L-pipecolic acid hydroxylase of the present invention is notlimited to a living microorganism or cell but includes those apparentlydead as living bodies and retaining the ability of the enzyme.

Moreover, the types of microorganisms or cells having the ability toproduce an L-pipecolic acid hydroxylase include those described below as“host microorganisms” or “host cells”.

Known methods such as recombinant gene technology (transformation) andmutagenesis may be employed as the means to impart the above productionability by breeding. Several different methods may be used as the methodof transformation, in which the expression of a gene of interest on DNAis enhanced by introducing DNA carrying the gene of interest, bymodifying an expression regulatory sequence of the gene, such as apromoter, on the chromosome, or the like.

Among them, it is preferred to use a microorganism or cell which hasbeen transformed with a DNA encoding the L-pipecolic acid hydroxylase ofthe present invention.

For example, a DNA encoding the L-pipecolic acid hydroxylase of thepresent invention is operatively inserted into a known expression vectorto construct an L-pipecolic acid hydroxylase-expressing vector, and theexpression vector is used to transform a host cell. Thus, a transformantinto which the DNA encoding the L-pipecolic acid hydroxylase of thepresent invention has been introduced can be produced.

Moreover, the transformant can also be produced by incorporation of theDNA encoding the L-pipecolic acid hydroxylase of the present inventioninto a chromosome of the host cell in such a way that expression of theenzyme is possible, which incorporation is mediated by a procedure suchas homologous recombination or the like.

The procedure for establishing the transformant, the method ofconstructing a recombinant vector suitable for the host, and the methodof culturing the host can be carried out in accordance with techniquesconventionally used in the fields of molecular biology, bioengineeringand genetic engineering (for example, methods described in MolecularCloning, supra).

Examples of the method of preparing the transformant include a method inwhich the DNA encoding the L-pipecolic acid hydroxylase of the presentinvention is introduced into a plasmid vector, phage vector, or virusvector that stably exists in a host cell and then the constructedexpression vector is introduced into the host cell, or a method in whichthe DNA is directly introduced into the host genome, in both of whichmethods the genetic information on the DNA is then transcribed andtranslated.

In this process, it is preferable to link a promoter to the DNA on the5′-upstream side and it is more preferable to further link a terminatorto the DNA on the 3′-downstream side, which promoter and terminator aresuitable in the host. As long as the promoter and the terminator are apromoter and a terminator which are known to be functional in a host,they can be used and are not particularly limited. For example, vectors,promoters, and terminators described in “Fundamental Microbiology 8:Genetic Engineering, Kyoritsu Shuppan Co., Ltd.” can be used.

The host microorganism or host cell to be transformed is notparticularly limited as long as the host itself does not adverselyaffect the reaction of L-pipecolic acid.

Examples of the host microorganism include prokaryotes such as E. coli(bacteria of the genus Escherichia), bacteria of the genus Bacillus,bacteria of the genus Pseudomonas, coryneform bacteria, root-nodulebacteria, bacteria of the genus Lactobacillus, bacteria of the genusBrevibacillus, bacteria of the genus Anaerobiospirillum, bacteria of thegenus Actinobacillus, actinomycetes, and the like; and eukaryotesincluding fungi, such as yeasts and filamentous fungi, plants, animals,and the like. Among them, E. coli, yeasts, and coryneform bacteria arepreferable, and E. coli is particularly preferable.

Examples of the host microorganism include microorganisms as describedbelow:

bacteria belonging to the genera Escherichia, Bacillus, Pseudomonas,Corynebacterium, Rhizobiun, Lactobacillus. Brevibacillus,Anaerobiospirillum, Actinobacillus, Serratia, Brevibacterium,Streptococcus, and the like, whose host vector systems have beenestablished:

actinomycetes belonging to the genera Rhodococcus, Streptomyces, and thelike, whose host vector systems have been established;

yeasts belonging to the genera Saccharomyces, Kluyveromyces,Schizosaccharomyces, Zygosaccharomyces, Yarrowia, Trichosporon,Rhodosporidium, Hansenula, Pichia, Candida, and the like, whose hostvector systems have been established; and

fungi belonging to the genera Neurospora, Aspergillus, Cephalosporium,Trichoderma, and the like, whose host vector systems have beenestablished.

Examples of preferable host microorganisms and of a preferabletransformation procedure, vector, promoter, and terminator for eachmicroorganism will be described below. However, the present invention isnot limited thereto.

For the genus Escherichia, especially Escherichia coli, examples of theplasmid vector include plasmids of the pBR series, the pUC series, andthe like; and examples of the promoter include lac (β-galactosidase),frp (tryptophan operon), tac, trc (a fusion of lac and trp), the PL andPR promoters of phage lambda, promoters derived from T7 phage, and thelike. Moreover, examples of the terminator include terminators derivedfrom trpA, phages, and rrnB ribosomal RNA, and the like. Among thosepromoters, a promoter that allows inducible gene expression may also beused for the purpose of improving the expression efficiency. Forexample, in the case of the above lac promoter, gene expression can beinduced by addition of an inducer such as lactose orisopropyl-fl-D-thiogalactoside (IPTG).

For the genus Bacillus, examples of the vector can include plasmidsbased on pUB110, pC194, and the like, which may be integrated into achromosome. As the promoter and the terminator, those of genes forenzymes, such as alkaline protease, neutral protease, and α-amylase, canbe used.

For the genus Pseudomonas, examples of the vector can include generichost vector systems established in Pseudomonas putida, Pseudomonascepacia, and the like; the wide-host-range vector pKT240 (containinggenes required for autonomous replication and derived from RSF1010 andthe like), which is based on a plasmid involved in degradation oftoluene compounds, TOL plasmid (Gene, 26, 273-82 (1983)); and the like.

For the genus Brevibacterium, especially Brevibacterium lactofermentum,examples of the vector can include plasmid vectors such as pAJ43 (Gene,39, 281 (1985)). As the promoter and the terminator, various promotersand terminators used in E. coli can be used.

For the genus Corynebacterium, especially Corynebacterium glutamicum,examples of the vector include plasmid vectors such as pCS11(JP-57-183799 A) and pCB101 (Mol. Gen. Genet., 196, 175 (1984)).

For Saccharomyces, especially Saccharomyces cerevisiae, examples of thevector include plasmids of the YRp series, the YEp series, the YCpseries, and the YIp series, and the like. Moreover, examples ofpromoters and terminators which may be used include those of the genesfor various enzymes such as alcohol dehydrogenase,glyceraldehyde-3-phosphate dehydrogenase, acid phosphatase,β-galactosidase, phosphoglycerate kinase, and enolase.

For the genus Schizosaccharomyces, examples of the vector can includeplasmid vectors, such as the plasmid vectors derived fromSchizosaccharomyces pombe described in Mol. Cell. Biol., 6, 80 (1986).In particular, pAUR224 is commercially available from Takara Shuzo Co.,Ltd. and readily available.

In the genus Aspergillus, Aspergillus niger, Aspergillus oryzae and thelike are the best-studied species among molds, in which plasmids andintegration into the chromosome are applicable, and promoters forextracellularly secreted protease and amylase can be used (Trends inBiotechnology, 7, 283-287 (1989)).

Moreover, examples of the host cell include cells from animals, such asinsects (for example, silkworm) (Nature, 315, 592-594 (1985)), and fromplants such as rapeseed, maize, and potato. Also, E. coli cell-freeextract and cell-free proteins from wheat germ and the like may also beused. A system utilizing a synthesis system has been established and maypreferably be used.

Moreover, various host vector systems other than the above-describedsystems have also been established, and those systems may be used asappropriate.

In the present specification, a “processed product of the microorganismor cell having the ability to produce an L-pipecolic acid hydroxylase”refers to a product that is prepared by culturing the microorganism orcell having the ability to produce an L-pipecolic acid hydroxylase, andthen 1) treating the microorganism or cell with an organic solvent,surfactant, and the like, 2) freeze-drying the microorganism or cell, 3)immobilizing the microorganism or cell on a carrier and the like, 4)physically or enzymatically disrupting the microorganism or cell, or 5)separating an enzyme fraction as a crudely or finely purified materialfrom any of the products of the aforementioned treatments 1) to 4).

Examples of the organic solvent include methanol, ethanol, 1-propanol,2-propanol, 1-butanol, tert-butanol, acetone, dimethyl sulfoxide, ethylacetate, butyl acetate, toluene, chloroform, n-hexane, and the like.

Examples of the surfactant include nonionic surfactants, such asTween-20, Triton X-100, Briji 35, dodecyl-β-D-maltoside, Nonidet P-40,and octyl-β-D-glucoside; amphoteric-ionic surfactants, such as3-(3-cholamidopropyl)dimethylammonio-1-propanesulphonate (CHAPS);anionic surfactants, such as sodium dodecyl sulphate (SDS); and thelike.

Specific examples of a procedure to immobilize the microorganism or cellon a carrier and the like include procedures to immobilize an enzymefraction from the microorganism or cell on a carrier, and otherprocedures, where representative examples of the carrier includepolyacrylamide gel, carrageenan gel, ion exchange resins, and the like.

Examples of a procedure to physically disrupt the microorganism or cellinclude sonication, high-pressure treatment with French Press,mechanical milling treatment using a homogenizer, Dyno-Mill bead mill,or the like, and other procedures.

Examples of a procedure to enzymatically disrupt the microorganism orcell include a procedure based on the treatment with an enzyme havingthe cell wall lysis activity, such as lysozyme or the like.

As a separation procedure in the enzyme purification, a conventionalprocedure for enzyme isolation and purification may be used. Forexample, a purified material can be separated from the extract preparedby any of the aforementioned procedures or the like, using each or acombination of centrifugation, ultrafiltration, salting-out withammonium sulfate or the like, desalting, precipitation with an organicsolvent, chromatography using various resins (anion exchangechromatography, cation exchange chromatography, hydrophobicchromatography, gel filtration, affinity chromatography),electrophoresis such as isoelectric focusing or the like, and the like.

Examples of the culture liquid comprising the L-pipecolic acidhydroxylase of the present invention and obtained by culturing themicroorganism or cell having the ability to produce the enzyme include asuspension of the microorganism or cell and a liquid medium; and incases where the microorganism or cell is a microorganism or cell forsecretory expression, the supernatant obtained by removing themicroorganism or cell by centrifugation and the like, and a concentrateof the supernatant; and the like.

In the present specification, a “culture liquid comprising theL-pipecolic acid hydroxylase and obtained by culturing the microorganismor cell having the ability to produce the enzyme” refers to 1) a cultureliquid of the microorganism or cell, 2) a culture liquid obtained bytreating the culture liquid of the microorganism or cell with an organicsolvent, a surfactant, and the like, and 3) a culture liquid in whichthe cell membrane of the microorganism or cell has been physically orenzymatically disrupted.

As the organic solvent, surfactant, and the physical or enzymaticdisruption method, the above-described materials and procedures can beused.

The method of producing a hydroxy-L-pipecolic acid according to thepresent invention is characterized by allowing an L-pipecolic acidhydroxylase, a microorganism or cell having the ability to produce theenzyme, a processed product of the microorganism or cell, and/or aculture liquid comprising the enzyme and obtained by culturing themicroorganism or cell to act on the L-pipecolic acid as the substrate inthe presence of 2-oxoglutaric acid and ferrous ion, and therebyproducing a hydroxy-L-pipecolic acid.

In the present invention, as the L-pipecolic acid as the substrate, pureL-pipecolic acid may be used and L-pipecolic acid containing a traceamount of L-proline may also be used. The content of L-proline isnormally not more than 10% (w/w), preferably not more than 1% (w/w), andparticularly preferably not more than 0.1% (w/w).

Moreover, as the L-pipecolic acid as the substrate, a commercialL-pipecolic acid may be used and an L-pipecolic acid biologicallyproduced by using an enzyme, microbial bodies, and the like may also beused. Biologically produced L-pipecolic acid may be contaminated withL-proline, for example, as a carry-over component from a medium used forthe preparation of the enzyme or the microbial bodies; in the presentinvention, L-pipecolic acid can be contaminated with the above-describedcontent of L-proline.

Examples of a method of biologically producing L-pipecolic acid includethe methods as described below:

(i) allowing L-lysine and/or DL-lysine to react with

-   -   (i-1) one or more enzymes selected from the group consisting of        an L-amino acid oxidase, an L-amino acid dehydrogenase and an        L-amino acid aminotransferase, or    -   (i-2) an amino acid racemase and one or more enzymes selected        from the group consisting of a D-amino acid oxidase, a D-amino        acid dehydrogenase and a D-amino acid aminotransferase,

for the production of 3,4,5,6-tetrahydropyridine-2-carboxylic acid; andsubsequently

allowing an N-methyl-L-amino acid dehydrogenase to act on the3,4,5,6-tetrahydropyridine-2-carboxylic acid for the production ofL-pipecolic acid;

(ii) allowing L-lysine to react with one or more enzymes selected fromthe group consisting of an L-lysine 6-oxidase, an L-lysine6-dehydrogenase and an L-lysine 6-aminotransferase, for the productionof 2,3,4,5-tetrahydropyridine-2-carboxylic acid; and subsequently

allowing a pyrroline-5-carboxylate reductase to act on the2,3,4,5-tetrahydropyridine-2-carboxylic acid for the production ofL-pipecolic acid; and

(iii) allowing a lysine cyclodeaminase to act on L-lysine for theproduction of L-pipecolic acid.

The first method of producing L-pipecolic acid in the present inventionis a production method as described below, the production methodcharacterized by:

(i) allowing L-lysine and/or DL-lysine to react with

-   -   (i-1) one or more enzymes selected from the group consisting of        an L-amino acid oxidase, an L-amino acid dehydrogenase and an        L-amino acid aminotransferase, or    -   (i-2) an amino acid racemase and one or more enzymes selected        from the group consisting of a D-amino acid oxidase, a D-amino        acid dehydrogenase and a D-amino acid aminotransferase,

for the production of 3,4,5,6-tetrahydropyridine-2-carboxylic acid; andsubsequently

allowing an N-methyl-L-amino acid dehydrogenase to act on the3,4,5,6-tetrahydropyridine-2-carboxylic acid for the production ofL-pipecolic acid.

The method will be described below by illustrating a scheme.

In Scheme (i-1), at least one enzyme selected from the group consistingof an L-amino acid oxidase, an L-amino acid dehydrogenase and an L-aminoacid transferase, and an N-methyl-L-amino acid dehydrogenase (NMAADH)are used.

First, the compound (a) (L-lysine) is converted to the compound (b) byan enzyme selected from the group consisting of an L-amino acid oxidase,an L-amino acid dehydrogenase and an L-amino acid transferase. Thecompound (b) is spontaneously converted to the compound (c)(3,4,5,6-tetrahydropyridine-2-carboxylic acid). Then, the compound (c)is converted to the compound (d) (L-pipecolic acid) by anN-methyl-L-amino acid dehydrogenase (NMAADH, also known as DpkA).

Here, the L-amino acid oxidase is not particularly limited as long as itis an enzyme which can catalyze the substitution of the amino group atposition 2 of L-lysine with an oxo group, but examples of the L-aminoacid oxidase include a protein as described in J. Biochem., 2015, 157(4), pp. 201, or proteins each having an amino acid sequence with anidentity of not less than 80%, preferably not less than 90%, and morepreferably not less than 95% to the amino acid sequence of the aboveprotein and maintaining the activity thereof.

The L-amino acid dehydrogenase is not particularly limited as long as itis an enzyme which can catalyze the substitution of the amino group atposition 2 of L-lysine with an oxo group, but examples of the L-aminoacid dehydrogenase include a protein as described in Nature, 1966, 211,pp. 854, or proteins each having an amino acid sequence with an identityof not less than 80%, preferably not less than 90%, and more preferablynot less than 95% to the amino acid sequence of the above protein andmaintaining the activity thereof.

The L-amino acid transferase (L-amino acid aminotransferase) is notparticularly limited as long as it is an enzyme which can catalyze thesubstitution of the amino group at position 2 of L-lysine with an oxogroup, but examples of the L-amino acid transferase include a proteincontaining an amino acid sequence as described in Eur. J. Biochem.,1998, 254, pp. 347, or proteins each having an amino acid sequence withan identity of not less than 80%, preferably not less than 90%, and morepreferably not less than 95% to the amino acid sequence of the aboveprotein and maintaining the activity thereof.

The N-methyl-L-amino acid dehydrogenase is not particularly limited aslong as it is an enzyme which can catalyze the conversion of3,4,5,6-tetrahydropyridine-2-carboxylic acid to L-pipecolic acid, butexamples of the N-methyl-L-amino acid dehydrogenase include DpkAdescribed in J. Biol. Chem. 2005, 280(49), pp. 40875, or proteins eachhaving an amino acid sequence with an identity of not less than 80%,preferably not less than 90%, and more preferably not less than 95% tothe amino acid sequence of DpkA and maintaining the activity thereof.

In Scheme (i-2), at least one enzyme selected from the group consistingof a D-amino acid oxidase, a D-amino acid dehydrogenase and a D-aminoacid transferase, an amino acid racemase, and an N-methyl-L-amino aciddehydrogenase (NMAADH) are used.

First, the compound (a) (L-lysine) is converted to the compound (a′) inthe D-form (D-lysine) by an amino acid racemase, which is then convertedto the compound (b) by an enzyme selected from the group consisting of aD-amino acid oxidase, a D-amino acid dehydrogenase and a D-amino acidtransferase. The compound (b) is spontaneously converted to the compound(c). Then, the compound (c) is converted to the compound (d)(L-pipecolic acid) by an N-methyl-L-amino acid dehydrogenase (NMAADH).

The D-amino acid oxidase is not particularly limited as long as it is anenzyme which catalyze the substitution of the amino group at position 2of D-lysine with an oxo group, but examples of the D-amino acid oxidaseinclude a protein containing an amino acid sequence as described inBiochemistry, 2005, 70, pp. 40, or proteins each having an amino acidsequence with an identity of not less than 80%, preferably not less than90%, and more preferably not less than 95% to the amino acid sequence ofthe above protein and having the activity thereof.

The D-amino acid dehydrogenase is not particularly limited as long as itis an enzyme which catalyze the substitution of the amino group atposition 2 of D-lysine with an oxo group, but examples of the D-aminoacid dehydrogenase include DauA described in Microbiology, 2010, 156 (Pt1), pp. 60 and Proc. Natl. Acad. Sci. U.S.A., 2009, 106, pp. 906, orproteins each having an amino acid sequence with an identity of not lessthan 80%, preferably not less than 90%, and more preferably not lessthan 95% to the amino acid sequence of DauA and maintaining the activitythereof.

The D-amino acid transferase (D-amino acid aminotransferase) is notparticularly limited as long as it is an enzyme which catalyze thesubstitution of the amino group at position 2 of D-lysine with an oxogroup, but examples of the D-amino acid transferase include D-AATdescribed in Protein Eng, 1998, 11, pp. 53, or proteins each having anamino acid sequence with an identity of not less than 80%, preferablynot less than 90%, and more preferably not less than 95% to the aminoacid sequence of D-AAT and maintaining the activity thereof.

The amino acid racemase (LysR) is not particularly limited as long as itis an enzyme which can catalyze the conversion of L-lysine to D-lysine,but examples of the amino acid racemase include LysR described in Appl.Microbiol. Biotechnol. 2015, 99, pp. 5045, or proteins each having anamino acid sequence with an identity of not less than 80%, preferablynot less than 90%, and more preferably not less than 95% to the aminoacid sequence of LysR and maintaining the activity thereof.

The N-methyl-L-amino acid dehydrogenase is not particularly limited aslong as it is an enzyme which can catalyze the conversion of3,4,5,6-tetrahydropyridine-2-carboxylic acid to L-pipecolic acid, butexamples of the N-methyl-L-amino acid dehydrogenase include DpkAdescribed in J. Biol. Chem. 2005, 280(49). pp. 40875, or proteins eachhaving an amino acid sequence with an identity of not less than 80%,preferably not less than 90%, and more preferably not less than 95% tothe amino acid sequence of DpkA and maintaining the activity thereof.

In the reactions indicated by Schemes (i-1) and (i-2), each step of theenzyme reaction may be separately performed, but it is preferred tosequentially perform the enzyme reaction steps in the same reactionsystem.

In cases where the above method is carried out continuously in the samereaction system, the method is preferably carried out in an aqueousmedium containing L-lysine and cells transformed with a group of genesencoding the respective enzymes, products prepared from thetransformants, and/or culture liquids obtained by culturing thetransformants, or alternatively in a mixture of the aqueous medium andan organic solvent.

Because N-methyl-L-amino acid dehydrogenase (NMAADH) requires NAD(P)H asa coenzyme, it is preferred to allow a system for the regeneration ofNAD(P)H from NAD(P)+, which is generated in the reaction of NMAAH, tocoexist in the same reaction system.

Exemplary methods for regeneration of NAD(P)H include 1) a method inwhich the ability of a host microorganism itself to reduce NAD(P)⁺ isutilized; 2) a method in which a microorganism having the ability togenerate NAD(P)H from NAD(P) or a product prepared therefrom, or anenzyme available for the regeneration of NAD(P)H (regeneration enzyme),such as glucose dehydrogenase, formate dehydrogenase, alcoholdehydrogenase, an amino acid dehydrogenase, or an organic aciddehydrogenase (such as malate dehydrogenase), is added to the reactionsystem; 3) a method in which genes for the above regeneration enzymesavailable for the regeneration of NAD(P)H are introduced together withthe DNA according to the present invention into a host; and the like.

Among them, in the above-described regeneration method (1), compoundssuch as glucose, ethanol, formic acid, and the like are preferably addedto the reaction system. Thus, these compounds are metabolized by a host,and NAD(P)H generated in the metabolic processes can be used in thereaction.

In the above-described regeneration method (2), as the regenerationenzymes, microorganisms containing those regeneration enzymes, orproducts prepared from microbial bodies of those microorganisms, such asthose prepared by acetone treatment, freeze-drying, and physical orenzymatic disruption, may be used. Moreover, fractions containing theenzymes separated as crudely or finely purified materials from theproducts prepared from the microbial bodies, those immobilized on acarrier such as polyacrylamide gel or carrageenan gel, and the like maybe used. Furthermore, commercially available products of the enzymes maybe used.

In the above-described regeneration methods (2) and (3), a compound as asubstrate for any of the regeneration enzymes is preferably added toallow the regeneration enzyme to exert its ability to reduce thecoenzyme. For example, glucose, formic acid, and an alcohol such asethanol or isopropanol are preferably added when a glucosedehydrogenase, a formic acid dehydrogenase, and an alcohol dehydrogenaseare used, respectively.

As the cells containing the enzymes which catalyze the respectivereaction steps, microorganisms inherently having these enzymes may beused, but it is preferred to use cells transformed with DNAs encodingthe respective enzymes. In Scheme (i-1), it is preferred to use cellstransformed with different DNAs encoding at least one enzyme selectedfrom the group consisting of an L-amino acid oxidase, an L-amino aciddehydrogenase and an L-amino acid transferase, and an N-methyl-L-aminoacid dehydrogenase (NMAADH). Moreover, in Scheme (i-2), it is preferredto use cells transformed with different DNAs encoding at least oneenzyme selected from the group consisting of a D-amino acid oxidase, aD-amino acid dehydrogenase and a D-amino acid transferase, an amino acidracemase, and an N-methyl-L-amino acid dehydrogenase (NMAADH).

Moreover, each of these DNAs may be integrated into a chromosome, orthese DNAs may be introduced into a single vector for use intransformation of a host, or each of these DNAs may be separatelyintroduced into a vector for use in subsequent transformation of a host.

The transformation method for a host cell, such as a microorganism, thetype of the host, and the like are the same as those as described abovefor the L-pipecolic acid hydroxylase.

The second method of producing L-pipecolic acid from L-lysine in thepresent invention is a production method as described below, theproduction method characterized by:

(ii) allowing L-lysine to react with one or more enzymes selected fromthe group consisting of an L-lysine 6-oxidase, an L-lysine6-dehydrogenase and an L-lysine 6-aminotransferase, for the productionof 2,3,4,5-tetrahydropyridine-2-carboxylic acid; and subsequently

allowing a pyrroline-5-carboxylate reductase to act on the2,3,4,5-tetrahydropyridine-2-carboxylic acid for the production ofL-pipecolic acid.

The method will be described below by illustrating a scheme.

First, the compound (a) (L-lysine) is converted to the compound (b′) byat least one enzyme selected from the group consisting of an L-lysine6-oxidase, an L-lysine 6-dehydrogenase and an L-lysine 6-transferase.The compound (b′) is spontaneously converted to the compound (c′)(2,3,4,5-tetrahydropyridine-2-carboxylic acid). Then, the compound (c′)is converted to the compound (d) (L-pipecolic acid) by apyrroline-5-carboxylate (P5c) reductase.

The L-lysine 6-oxidase is not particularly limited as long as it is anenzyme which can catalyze the substitution of the amino group atposition 6 of L-lysine with an oxo group, but examples of the L-lysine6-oxidase include LodA described in Biochim. Biophys. Acta., 2006, 1764,pp. 1577, or proteins each having an amino acid sequence with anidentity of not less than 80%, preferably not less than 90%, and morepreferably not less than 95% to the amino acid sequence of LodA andmaintaining the activity thereof.

The L-lysine 6-dehydrogenase is not particularly limited as long as itis an enzyme which can catalyze the substitution of the amino group atposition 6 of L-lysine with an oxo group, but examples of the L-lysine6-dehydrogenase include a protein containing an amino acid sequence asdescribed in J. Biochem., 105, pp. 1002-1008 (1989), or proteins eachhaving an amino acid sequence with an identity of not less than 80%,preferably not less than 90%, and more preferably not less than 95% tothe amino acid sequence of the above protein and having the activitythereof.

The L-lysine 6-transferase (lysine-6-aminotransferase) is notparticularly limited as long as it is an enzyme which can catalyze thesubstitution of the amino group at position 6 of hydroxy-L-lysine withan oxo group, but examples of the L-lysine 6-transferase include aprotein containing an amino acid sequence as described in WO2001/048216,or proteins each having an amino acid sequence with an identity of notless than 80%, preferably not less than 90%, and more preferably notless than 95% to the amino acid sequence of the above protein and havingthe activity thereof.

The pyrroline-5-carboxylate (P5C) reductase is not particularly limitedas long as it is an enzyme which can catalyze the conversion of2,3,4,5-tetrahydropyridine-2-carboxylic acid to L-pipecolic acid, butexamples of the pyrroline-5-carboxylate (P5C) reductase include aprotein containing an amino acid sequence as described in WO2001/048216,or proteins each having an amino acid sequence with an identity of notless than 80%, preferably not less than 90%, and more preferably notless than 95% to the amino acid sequence of the above protein and havingthe activity thereof.

In the reaction of the production method for pipecolic acid (ii), eachstep of the enzyme reaction may be separately performed, but it ispreferred to sequentially perform the enzyme reaction steps in the samereaction system. In particular, it is preferred to perform the reactionby allowing cells containing the enzymes, which catalyze the respectivereaction steps, to react with L-lysine. As the cells containing theenzymes which catalyze the respective reaction steps, cells inherentlyhaving these enzymes may be used, but it is preferred to use cellstransformed with DNAs encoding the respective enzymes. Specifically, itis preferred to use cells transformed with different DNAs encoding atleast one enzyme selected from the group consisting of an L-lysine6-oxidase, an L-lysine 6-dehydrogenase and an L-lysine 6-transferase,and a pyrroline-5-carboxylate (P5C) reductase.

The transformation method for a host cell, such as a microorganism, thetype of the host, and the like are the same as those as described abovefor the L-pipecolic acid hydroxylase.

In cases where the second method (ii) is carried out continuously in thesame reaction system, the method is preferably carried out in an aqueousmedium containing L-lysine and cells transformed with a group of genesencoding the respective enzymes, products prepared from thetransformants, and/or culture liquids obtained by culturing thetransformants, or alternatively in a mixture of the aqueous medium andan organic solvent.

The third method of producing L-pipecolic acid from L-lysine in thepresent invention is a production method as described below, theproduction method characterized by:

(iii) allowing a lysine cyclodeaminase to act on L-lysine for theproduction of L-pipecolic acid.

The lysine cyclodeaminase is not particularly limited as long as it isan enzyme which can catalyze the conversion of L-lysine to L-pipecolicacid, but examples of the lysine cyclodeaminase include a proteincontaining an amino acid sequence as described in Biochimie 2007, 89,pp. 591, or proteins each having an amino acid sequence with an identityof not less than 80%, preferably not less than 90%, and more preferablynot less than 95% to the amino acid sequence of the above protein andhaving the activity thereof.

It is preferred to perform the reaction by the lysine cyclodeaminase byallowing cells containing a lysine cyclodeaminase to react withL-lysine. As the microorganism containing a lysine cyclodeaminase, cellsinherently having the enzymes may be used, but it is preferred to use acell transformed with a DNA encoding a lysine cyclodeaminase.

The transformation method for a host cell, such as a microorganism, thetype of the host, and the like are the same as those as described abovefor the L-lysine hydroxylase.

In cases where the conversion of L-lysine to L-pipecolic acid by alysine cyclodeaminase is performed, the conversion is preferablyperformed in an aqueous medium containing L-lysine and cells transformedwith a DNA encoding a lysine cyclodeaminase, a product prepared from thetransformant, and/or a culture liquid obtained by culturing thetransformant, or alternatively in a mixture of the aqueous medium and anorganic solvent.

In the present invention, the L-pipecolic acid hydroxylase, themicroorganism or cell having the ability to produce the enzyme, theprocessed product of the microorganism or cell, or the culture liquidcomprising the enzyme and obtained by culturing the microorganism orcell can be contaminated with L-proline. In that case, the content ofL-proline is not more than 10% (w/w), preferably not more than 1% (w/w),and particularly preferably not more than 0.1% (w/w).

Moreover, the microorganism or cell having the ability to produce anL-pipecolic acid hydroxylase may be a microorganism or cell having theability to produce L-proline from an ingredient as a base material inthe culture liquid or the reaction system.

Moreover, a solvent may be used when a hydroxy-L-pipecolic acid isproduced. The solvent is not particularly limited, but the production ofa hydroxy-L-pipecolic acid is preferably carried out in an aqueoussolvent, or in a mixture of an aqueous solvent and an organic solvent.

Examples of the aqueous medium include water or buffers. Examples of thebuffer include phosphate buffer, acetate buffer, borate buffer, Tris-HClbuffer, and the like.

Moreover, as an organic solvent to be used in the mixture, an organicsolvent such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol,tert-butanol, acetone, or dimethyl sulfoxide, in which the solubility ofthe reaction substrate is high, may be used, while an organic solventsuch as ethyl acetate, butyl acetate, toluene, chloroform, or n-hexane,which is effective in the removal of reaction by-products, and the like,may also be used.

The volume of the solvent is not particularly limited, but it is avolume normally in the range of 0.01% (w/v) to 90% (w/v), and preferablyin the range of 0.1% (w/v) to 10% (w/v), to the L-pipecolic acid as thesubstrate.

The concentration of the L-pipecolic acid as the substrate in thereaction system is normally in the range of 0.01% (w/v) to 90% (w/v),and preferably in the range of 0.1% (w/v) to 30% (w/v). The L-pipecolicacid may be added in one portion to the reaction system when thereaction is initiated, but is desirably added to the reaction system ina continuous or intermittent fashion in view of the inhibitory effect onthe substrate of the enzyme, if any to be reduced, the increase in theconcentration of the accumulated product, and the like.

The amount of 2-oxoglutaric acid to be added is an amount equimolar toor greater than that of the L-pipecolic acid as the substrate, and ispreferably an amount ranging from 1 to 1.2 times the number of moles ofthe L-pipecolic acid as the substrate. The 2-oxoglutaric acid may beadded in one portion to the reaction system when the reaction isinitiated, but is desirably added to the reaction system in a continuousor intermittent fashion in view of the inhibitory effect on thesubstrate of the enzyme, if any, to be reduced, the increase in theconcentration of the accumulated product, and the like.

Moreover, an inexpensive compound that can be metabolized by the host,such as glucose, may be added instead of 2-oxoglutaric acid to thereaction system, which compound is metabolized by the host and theresulting 2-oxoglutaric acid produced during this process may be allowedto exist in the reaction system.

The concentration of ferrous ion in the reaction system normally from0.0001 to 0.5 mol, preferably from 0.001 to 0.1 mol, per mol ofL-pipecolic acid.

The ferrous ion may be added in one portion, normally in the form offerrous sulfate, ferrous chloride, ferrous citrate, and the like, to thereaction system when the reaction is initiated. When the added ferrousions are oxidized to ferric ions or precipitated during the reaction andthe level of ferrous ions is thus decreased, it is also effective to addan additional amount of ferrous ion until a desired concentration isachieved.

Moreover, in cases where ferrous ions have already been contained at adesired concentration in the L-pipecolic acid hydroxylase of the presentinvention, the microorganism or cell having the ability to produce theenzyme, the processed product of the microorganism or cell, and/or theculture liquid comprising the enzyme and obtained by culturing themicroorganism or cell, the addition of ferrous ion is not necessarilyrequired.

The reaction is carried out at a reaction temperature of normally 4° C.to 60° C., preferably 15° C. to 45° C., and particularly preferably 20°C. to 40° C., and at a pH of normally 3 to 11, preferably 5 to 8. Thereaction time is normally about 1 hour to about 150 hours.

The L-pipecolic acid hydroxylase, the microorganism or cell having theability to produce the enzyme, the processed product of themicroorganism or cell, and/or the culture liquid comprising the enzymeand obtained by culturing the microorganism or cell are/is in such anamount that, for example, the concentration of the microorganism or cellin the reaction system is normally in the range of 0.1% (w/v) to 50%(w/v), preferably 1% (w/v) to 20% (w/v), in terms of wet cell weight ifthe microorganism or cell is used. Moreover, if the processed product orthe culture liquid is used, the processed product or the culture liquidis used in an amount based on the calculated specific activity of theenzyme, which corresponds to the concentration of the microorganism orcell in the reaction system as described above.

Produced hydroxy-L-pipecolic acids can be purified as follows: after thecompletion of the reaction, microbial bodies, proteins and the like inthe reaction system are separated by centrifugation, membranefiltration, and the like; a hydroxy-L-pipecolic acid is subsequentlypurified by an appropriate combination of extraction with an organicsolvent, such as 1-butanol, tert-butanol, or like; distillation; columnchromatography using an ion exchange resin, silica gel and the like;isoelectric precipitation; crystallization with a salt, such asmonohydrochloride salts, dihydrochloride salts and calcium salts; andthe like.

EXAMPLES

Now, the present invention will be described in more detail by way ofexamples but is not limited thereto.

<Example 1> Cloning of L-Pipecolic Acid Hydroxylase Genes

A gene encoding the amino acid hydroxylase McPH (GenBank Accession No.WP_030487089; SEQ ID NO: 2), which is annotated as an aspartylbeta-hydroxylase derived from a Micromonospora chokoriensis strain, wasartificially synthesized as a gene sequence codon-optimized forexpression in E. coli (mcph_OE; SEQ ID NO: 1). The gene was insertedinto pJExpress411 (DNA2.0) to construct the plasmid pJ411McPH.

Similarly, four genes for enzymes exhibiting the activity to catalyzethe hydroxylation of L-pipecolic acid at position 5 were cloned.

The codon-optimized gene sequences sruph_OE (SEQ ID NO: 3), caph_OE (SEQID NO: 5), and xdph_OE (SEQ ID NO: 7) for expression in E. coli, whichrespectively encode the L-pipecolic acid cis-5-hydroxylase SruPH(GenBank Accession No. EFV12517; SEQ ID NO: 4) derived from theSegniliparus rugosus strain ATCC BAA-974, the L-prolinecis-4-hydroxylase CaPH (GenBank Accession No. WP_012787640; SEQ ID NO:6) derived from the Catenulispora acidiphila strain NBRC 102108, and theL-proline cis-4-hydroxylase XdPH (GenBank Accession No. CDGl16639; SEQID NO: 8) derived from the Xenorhabdus doucetiae strain FRM16, wereartificially synthesized.

The respective genes were inserted into pJexpress411 (manufactured byDNA2.0) and obtained as plasmids, which were named pJ411 SruPH,pJ411CaPH, and pJ411XdPH.

The codon-optimized gene sequence smph_OE (SEQ ID NO: 9) for expressionin E. coli, which encodes the L-proline cis-4-hydroxylaseSmPH (GenBankAccession No. CAC47686; SEQ ID NO: 10) derived from the Sinorhizobiummeliloti strain 1021, was artificially synthesized and inserted intopJexpress401 to produce the plasmid pJ401SmPH. For the smph_OE, theprimers smph_f (SEQ ID NO: 21) and smph_r (SEQ II) NO: 22) weresynthesized and used for PCR reaction using the plasmid DNA as atemplate according to a conventional method to obtain a DNA fragmentwith a length of about 1.0 kbp. The obtained DNA fragment was digestedwith the restriction enzymes Nde I and Hind III and then ligatedaccording to a conventional method into pET24a (Novagen) digested withNde I and Hind III to obtain pEI24SmPH.

A gene encoding the amino acid hydroxylase KjPH (GenBank Accession No.WP_046758372; SEQ ID NO: 18), which is annotated as a hypotheticalprotein derived from a Kordia jejudonensis strain, was artificiallysynthesized as a gene sequence codon-optimized for expression in E. coli(kjph_OE; SEQ ID NO: 17). The gene was inserted into pJExpress411(DNA2.0) to construct the plasmid pJ411KjPH.

Moreover, three genes for enzymes exhibiting the activity to catalyzethe hydroxylation of L-pipecolic acid at position 4 were cloned.

The codon-optimized gene sequences cgph_OE (SEQ ID NO: 11), pcph_OE (SEQID NO: 13), and gzph_OE (SEQ ID NO: 15) for expression in E. coli, whichrespectively encode the L-pipecolic acid trans-4-hydroxylase CgPH(GenBank Accession No. ELA34460; SEQ ID NO: 12) derived fromColletotrichum gloeosporioides strain Nara qc5, the L-pipecolic acidtrans-4-hydroxylase PcPH (GenBank Accession No. XP_002558179: SEQ ID NO:14) derived from the Penicillium chrysogenum strain Wisconsin 54-1255,and the L-pipecolic acid trans-4-hydroxylase GzPH (GenBank Accession No.XP 383389; SEQ II) NO: 16) derived from the Gibberella zeae strain PH-1,were artificially synthesized.

Details about the above-described cloned L-pipecolic acid hydroxylasesare shown in Table 1.

TABLE 1 SEQ ID NO. ID Microorganism of origin Accession No. 2 McPHMicromonospora chokoriensis WP_030487089 4 SruPH Segniliparus rugosusATCC EFV12517 BAA-974 6 CaPH Catenulispora acidiphila WP_012787640NBRC102108 8 XdPH Xenorhabdus doucetiae FRM16 CDG16639 10 SmPHSinorhizobium meliloti 1021 CAC47686 12 CgPH Colletotrichumgloeosporioides ELA34460 Nara gc5 14 PcPH Penicillium chrysogenumXP_002558179 Wisconsin 54-1255 16 GzPH Gibberella zeae PH-1 XP_383389 18KjPH Kordia jejudonensis WP_046758372

<Example 2> Obtainment of L-Pipecolic Acid Hydroxylase Gene-ExpressingBacteria

Each of the plasmids obtained in Example 1 was used to transform E. coli(Escherichia coli) BL21 (DE3) (manufactured by Invitrogen) according toa conventional method and thus to obtain the recombinant E. coli BL21(DE3)/pJ411McPH, BL21 (DE3)/pJ411SruPH, BL21 (DE3)/pJ411 CaPH, BL21(DE3)/pJ411XdPH, BL21 (DE3)/pET24SmPH, BL21 (DE3)/pJ411CgPH, BL21(DE3)/pJ411PcPH, BL21 (DE3)/pJ411GzPH, and BL21 (DE3)/pJ411KjPH.

To obtain bacteria expressing each of the introduced genes, eachrecombinant E. coli strain was cultured using liquid LB mediumcontaining kanamycin and a lac promoter-inducible substance for aboutfive hours at 30° C. and then for about 30 hours at 18° C., followed byharvest of the bacteria.

Each of the obtained recombinant E. coli strains in a volume of 0.6 mLwas recovered by centrifugation and was suspended in 0.5 mL of 50 mmol/LMES (2-morpholinoethanesulfonic acid) buffer at pH 6. The containercontaining the suspension was placed in ice-water bath to performsonication and then spun at 12,000 rpm. The obtained supernatant wasused as an enzyme solution in Example 3.

<Example 3> Determination of the Catalytic Efficiency (Kcat/Km) of theL-Pipecolic Acid Hydroxylase with L-Pipecolic Acid and L-Proline

Several L-pipecolic acid hydroxylases were examined for the catalyticefficiency with L-pipecolic acid and L-proline.

To a plastic tube, L-pipecolic acid at a final concentration rangingfrom 0.3 mmol L to 50 mmol/L, 2-oxoglutaric acid at 20 mmol/L,L-ascorbic acid at 1 mmol/L, ferrous sulfate (II) at 0.5 mmol/L, andeach enzyme solution obtained in Example 2 at a protein concentration ofabout 2 mg/mL were added; then, the obtained reaction solution in avolume of 0.2 mL was shaken at 30° C. for 25 minutes. Subsequently, 0.05mL of 1 mol/L hydrochloric acid was added to stop the reaction.

The reaction using L-proline as a substrate was carried out under thesame conditions as in the above reaction using L-pipecolic acid.

Then, each reaction solution was treated using1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (FDAA) (manufactured by TokyoChemical Industry Co., Ltd.) according to a method as described below,to obtain a FDAA-derivative of a hydroxy-L-pipecolic acid orhydroxy-L-proline included in each reaction solution.

To the reaction solution after stopping the reaction, 0.05 mL of 1 mol/Lsodium hydroxide was added for neutralization. After centrifuging thesolution at 12,000 rpm, 15 μL of 0.5 mol/L borate buffer (pH 9) wasadded to 15 μL of the obtained supernatant, followed by further additionof 30 μL of 20 mmol/L FDAA solution in acetone and incubation at 40° C.for one hour. Subsequently, 10 μL of 1 mol/L hydrochloric acid was addedto stop the derivatization reaction. The resulting solution was dilutedby adding 80 μL of methanol, and then centrifuged at 12,000 rpm. Theobtained supernatant was used as a FDAA derivative solution.

The amount of produced hydroxy-L-pipecolic acid or hydroxy-L-proline wasanalyzed with UPLC-MS (manufactured by Waters Co.). The analyticalconditions are shown in Tables 2 to 4.

The (5S)-hydroxy-L-pipecolic acid-producing activity or(4R)-hydroxy-L-pipecolic acid-producing activity (U/mg) indicated byeach enzyme solution was defined by units (U) per milligram of eachhydroxylase (mg). One unit herein represents the ability to produce 1μmol of a hydroxy-L-pipecolic acid in the period of 1 minute.

TABLE 2 LC settings Used ACQUITY SQD UPLC/MS (manufactured by Watersinstruments Co.) Analytical ACQUITY UPLC BEH C18 Column(manufactured bycolumn Waters Co.) Column temp. 30° C. Eluent A 0.1% formic acid/watersolution B 0.1% formic acid/acetonitrile solution

TABLE 3 Elution conditions Time A B Flow rate (min) (%) (%) (ml/min) 080 20 0.2 12 45 55 12.5 0 100 14.5 0 100 14.6 80 20 18 80 20

TABLE 4 MS conditions Used instruments SQ Detector (manufactured byWaters Co.) Settings Ion mode ESI Positive Capillary Voltage (kV) 3.0Cone Voltage (V) 50 Extractor Voltage (V) 3 RF Lens Voltage (V) 0.1Source Temp. (° C.) 130 Desolvation Temp. (° C.) 350 Desolvation GasFlow (L/hr) 600 Cone Gas Flow (L/hr) 50

The amount of each hydroxylase was quantified as described below.

An aliquot containing 5 μg of total protein was taken from each enzymesolution obtained in Example 2, loaded on each lane, and thenelectrophoresed on polyacrylamide gel. The obtained electrophoretogramwas analyzed using the image analysis software Image Lab3.0(manufactured by Bio-Rad Laboratories, Inc.) to determine the content ofeach hydroxylase. A carbonic anhydrase (manufactured by Sigma-AldrichCo. LLC) was used as a quantification reference. The content of ahydroxylase of interest was calculated from the signal intensity of theenzyme of interest, based on the signal intensities of bandscorresponding to 200 ng, 400 ng and 600 ng of the quantificationreference provided for the electrophoresis.

The catalytic efficiency of each enzyme was calculated based on theMichaelis-Menten kinetic equation.

First, the reciprocals of the respective concentrations of L-pipecolicacid (mmol/L) and the reciprocals of the hydroxy-L-pipecolicacid-producing activities per milligram of each hydroxylase (U/mg) atthe respective concentrations of L-pipecolic acid were plotted on thex-axis and the y-axis, respectively, where there are four to six datapoints in the range of 0.3 mmol/L to 50 mmol/L of L-pipecolic acid (FIG.1). It is known that, in FIG. 1, the reciprocal of each intercept withthe x-axis, multiplied with −1, represents a km value (mmol/L) and thereciprocal of each intercept with the y-axis represents a Vmax value(U/mg), the maximum rate of reaction.

The catalytic ability per molecule of the enzyme (kcat) can becalculated from the Vmax and the molecular weight of the enzyme. Table 5indicates the result of the calculation of catalytic efficiency(kcat/km), which is an index representing the catalytic ability of anenzyme, based on the obtained kcat and km values.

When L-proline was used as a substrate, a much amount ofhydroxy-L-proline was detected from the reaction solution using thehydroxylase CaPH. Moreover, a small amount of hydroxy-L-proline wasdetected from the reaction solutions of the hydroxylases SruPH, XdPH,SmPH, and KjPH. On the other hand, no hydroxy-L-proline was detectedfrom the reaction solution of the hydroxylase McPH. Based on thisresult, it is understood that McPH is a novel enzyme having the5-hydroxypipecolic acid-producing activity and characterized by a verylow reactivity with proline. This property is not easily deduced fromknown facts.

Also, no hydroxy-L-proline was detected from the reaction solutions ofthe hydroxylases CgPH, PcPH, and GzPH. Based on this result, it wasfound that these hydroxylases were novel enzymes having the4-hydroxypipecolic acid-producing activity and characterized by a verylow reactivity with proline. This property is not easily deduced fromknown facts.

TABLE 5 A value of the catalytic reaction efficiency for L- Substrate:L-pipecolic acid Substrate: L-proline proline relative to the Vmax Vmaxcatalytic reaction Molecular Km (U/mg- kcat/ Km (U/mg- kcat/ efficiencyfor L- Name weight (mM) protein) kcat Km (mM) protein) kcat Km pipecolicacid (5-hydroxy-L-pipecolic acid-producing enzyme) McPH 31872 0.24 0.060.03 0.13 (No activity) 0 SruPH 33774 1.09 0.05 0.03 0.02 0.40 0.13 0.070.18 754% CsPH 32503 (No activity) 8.87 0.02 0.01 0.00 — XdPH 33475 0.301.14 0.64 2.16 0.31 1.44 0.80 2.58 119% SmPH 32019 0.31 0.03 0.02 0.060.55 0.06 0.03 0.06 114% KiPH 32277 0.49 0.30 0.16 0.33 0.48 0.34 0.160.38 115% (4-hydroxy-L-pipecolic acid-producing enzyme) CgPH 37320 8.5670.42 43.50 5.12 (No activity) 0 PcPH 32130 3.67 16.16 8.55 2.36 (Noactivity) 0 GzPH 37835 4.72 114.94 72.48 15.36 (No activity) 0

<Example 4> Comparison of the Reactivity Among L-Pipecolic Acid5-Hydroxylases

The L-pipecolic acid 5-hydroxylases were compared for the reactivitywith L-proline and L-pipecolic acid.

To a plastic tube, L-pipecolic acid or L-proline at 10 mmol/L,2-oxoglutaric acid at 20 mmol/L, L-ascorbic acid at 1 mmol/L, ferroussulfate at 0.5 mmol/L, and each enzyme solution obtained in Example 2 ata total protein concentration of about 2 mg/mL were added; then, theobtained reaction solution in a volume of 0.2 mL was shaken at 30° C.for 30 minutes. The amounts of produced hydroxy-L-pipecolic acid andhydroxy-L-proline were determined by the same method based on thederivatization with FDAA as in Example 3. The detection of the productswas based on the absorbance at a wavelength of 340 nm.

FIG. 2 shows chromatograms from the LC analysis of the reactionsolutions derivatized with FDAA. The activity values were evaluated bythe amount of a product per gram of total protein (U/g-protein). Thehydroxy-L-proline-producing activity (relative activity) of each sampleis presented in Table 6, where a ratio of 100% corresponds to thehydroxy-L-pipecolic acid-producing activity of the same reactionsolution.

As indicated in FIG. 2 and Table 6, a much amount of hydroxy-L-prolinewas detected from the reaction solution of the hydroxylase CaPH.Moreover, a small amount of hydroxy-L-proline was detected from thereaction solutions of the hydroxylases SruPH, XdPH, and SmPH. Nohydroxy-L-proline was detected from the reaction solution of thehydroxylase McPH.

Based on this result, it is understood that McPH is a novel enzymehaving a high 5-hydroxypipecolic acid-producing activity andcharacterized by a very low reactivity with proline. This property isnot easily deduced from known facts.

Moreover, the hydroxylases SruPH, XdPH, and SmPH were found to beenzymes having a high 5-hydroxypipecolic acid-producing activity andcharacterized by a low reactivity with proline. This property is noteasily deduced from known facts.

TABLE 6 Relative activity (A value of the hydroxy-L-proline-producingactivity, where a value of 100 corresponds to the hydroxy-L-pipecolicacid-producing activity) Enzyme Substrate: L-pipecolic acid Substrate:L-proline McPH 100 0 SruPH 100 59 CaPH 100 583 XdPH 100 37 SmPH 100 46

<Example 5> Production of Hydroxy-L-Pipecolic Acids

Eight recombinant E. coli strains obtained in Example 2, that is, BL21(DE3)/pJ411McPH, BL21 (DE3)/pJ411SruPH, BL21 (DE3)/pJ411CaPH, BL21(DE3)/pJ411XdPH, BL21 (DE3)/pET24SmPH, BL21 (DE3)/pJ411CgPH, BL21(DE3)/pJ411PcPH and BL21 (DE3)/pJ411GzPH were inoculated to liquid M9medium for starter culture (33.9 g/L Na₂HPO₄, 15 g/L KH₂PO₄, 2.5 g/Lsodium chloride, 5 g/L ammonium chloride, 10 g/l, casamino acid, 0.1mmol/L calcium chloride, 0.1 mmol/L ferrous sulfate, 4 g/L glucose,0.001 mmol/L magnesium chloride) containing kanamycin sulfate (25μg/mL), and cultured at 30° C. for 24 hours under shaking conditions at200 rpm.

Forty μL of the culture liquid was added to liquid M9 medium for mainculture (50.9 g/L Na₂HPO₄, 22.5 g/L KH₂PO₄, 3.8 g/L sodium chloride, 7.5g/L ammonium chloride, 10 g/L casamino acid, 0.1 mmol/L calciumchloride, 0.1 mmol/L ferrous sulfate, 20 mmol/L L-pipecolic acid, 12.5g/L glycerol) containing kanamycin sulfate (25 μg/mL) and OvernightExpress Autoinduction Systems (Merck KGaA), and then cultured at 30° C.for 120 hours under shaking conditions at 200 rpm.

Samples for analysis were prepared by collecting and centrifuging theculture 48, 64, and 120 hours after the start of culture, and thenrecovering the supernatants, and the prepared samples were subjected toLC and MS analyses.

The samples for analysis were treated using FDAA (manufactured by TokyoChemical Industry Co., Ltd.) according to a method as described below,to obtain a FDAA-derivative.

After centrifuging the sample solutions for analysis, 27 μL of 0.5 mol/Lborate buffer (pH 9) was added to 3 μL of each supernatant, followed byfurther addition of 30 μL of 20 mmol/L FDAA solution in acetone andincubation at 40° C. for one hour. Subsequently, 10 μL of 1 mol/Lhydrochloric acid was added to stop the reaction. The resulting solutionwas diluted by adding 80 μL of methanol, and then centrifuged at 12,000rpm. The obtained supernatant was used as a FDAA derivative solution.

The obtained FDAA derivative solution was analyzed for the amounts of ahydroxy-L-pipecolic acid and hydroxy-L-proline under LC/MS conditionsindicated in Table 2. The obtained result is shown in FIG. 3 and FIG. 4.

FIG. 3 and Table 7 indicate the result on productivity obtained when(5S)-hydroxy-L-pipecolic acid-producing enzymes are used. FIG. 3 showsthe temporal change in amount of accumulated (5S)-hydroxy-L-pipecolicacid. Table 7 shows the accumulation amount of each component determinedfor the samples of 120 hours post inoculation. It is indicated thatalmost the entire amount of the substrate was allowed to convert to(5S)-hydroxy-L-pipecolic acid by incubation with the hydroxylases SruPHand McPH for 120 hours.

TABLE 7 Accumulation amount of each component after 120 hours(5S)-Hydroxy- (4S)-hydroxy- Name of L-pipecolic acid L-pipecolic acidL-proline enzyme (mmol/L) (mmol/L) (mmol/L) McPH 23.5 1.8 0 SruPH 22.50.1 1.8 CaPH 0.8 18.8 0 XdPH 15.2 10.6 0.3 SmPH 9.4 5.6 0.2

Accumulation of (4S)-hydroxy-L-proline was confirmed in the hydroxylasesSruPH, XdPH, and SmPH, in which the reactivity with proline isidentified in Example 3. No accumulation of (4S)-hydroxy-L-proline wasconfirmed in McPH. Also, no accumulation of (4S)-hydroxy-L-proline isconfirmed in the hydroxylase CaPH; however, it is likely due to a smallcapacity of the hydroxylase CaPH as a hydroxylase because theaccumulated amount of (5S)-hydroxy-L-pipecolic acid is also small.

Based on these results, it is understood that the hydroxylase McPH is avery practical hydroxylase having a high productivity of(5S)-hydroxy-L-pipecolic acid and characterized by no accumulation ofby-products which are hardly removed during the purification steps, suchas (4S)-hydroxy-L-proline.

FIG. 4 and Table 8 indicate the result on productivity obtained when(4S)-hydroxy-L-pipecolic acid-producing enzymes are used. FIG. 4 showsthe temporal change in amount of accumulated (4S)-hydroxy-L-pipecolicacid. Table 8 shows the accumulation amount of each component determinedfor the samples of 64 hours post inoculation. It is indicated that amajor amount of the substrate was allowed to convert to(4S)-hydroxy-L-pipecolic acid by incubation with the hydroxylases CgPH,PcPH, and GzPH for 64 hours. Moreover, no accumulation of(4R)-hydroxy-L-proline was confirmed.

Based on these results, it is understood that the hydroxylases CgPH,PcPH, and GzPH are very practical hydroxylases having a highproductivity of (4S)-hydroxy-L-pipecolic acid and characterized by noaccumulation of by-products which are hardly removed during thepurification steps, such as (4R)-hydroxy-L-proline.

TABLE 8 Accumulation amount of each component after 64 hours(4S)-hydroxy- (4R)-hydroxy- Name of L-pipecolic acid L-pipecolic acidL-proline enzyme (mmol/L) (mmol/L) (mmol/L) CgPH 19.1 0.2 0 PcPH 17.42.1 0 GzPH 18.8 0.2 0

<Example 6> Modification of the Gene for the Hydroxylase McPH ThroughMutagenesis

A plasmid encoding a mutant (McPHm1) was constructed using, as atemplate, the plasmid pJ411McPH obtained in Example 1 as well as using aset of the primer represented by SEQ ID NO: 23 (H4Q-f) and the primerrepresented by SEQ ID NO: 24 (H4Q-r) indicated in the Sequence Listing,where the QuikChange Multi Site-Directed Mutagenesis Kit (manufacturedby Stratagene) was used to replace histidine with glutamine at aminoacid position 4.

Plasmids were constructed in the same manner as described above, where aset of the primer represented by SEQ ID NO: 25 (F5Y-f) and the primerrepresented by SEQ ID NO: 26 (F5Y-r), a set of the primer represented bySEQ ID NO: 27 (C23A-f) and the primer represented by SEQ ID NO: 28(C23A-r), a set of the primer represented by SEQ ID NO: 29 (C44A-f) andthe primer represented by SEQ ID NO: 30 (C44A-r), a set of the primerrepresented by SEQ ID NO: 31 (L97R-f) and the primer represented by SEQID NO: 32 (L97R-r), a set of the primer represented by SEQ ID NO: 33(V98A-f) and the primer represented by SEQ ID NO: 34 (V98A-r), a set ofthe primer represented by SEQ ID NO: 35 (D116G-f) and the primerrepresented by SEQ ID NO: 36 (D116G-r), a set of the primer representedby SEQ ID NO: 37 (C137A-f) and the primer represented by SEQ ID NO: 38(C137A-r), and a set of the primer represented by SEQ ID NO: 39(D282E-f) and the primer represented by SEQ ID NO: 40 (D282E-r) weredesigned and used to replace phenylalanine with tyrosine at position 5(McPHm2), cysteine with alanine at position 23 (McPHm3), cysteine withalanine at position 44 (McPHm4), leucine with arginine at position 97(McPHm5), valine with alanine at position 98 (McPHm6), aspartic acidwith glycine at position 116 (McPHm7), cysteine with alanine at position137 (McPHm8), and aspartic acid with glutamic acid at position 282(McPHm9), respectively.

Each of the obtained plasmids was used to transform E. coli (Escherichiacoli) BL21 (DE3) (manufactured by Invitrogen) according to aconventional method and thus to obtain recombinant E. coli strainsexpressing the respective mutants. Enzyme solutions were preparedaccording to the method described in Example 2 from the obtainedrecombinant E. coli strains and evaluated for the activity to catalyzethe hydroxylation of L-pipecolic acid at position 5 according to themethod described in Example 4. The activity values were evaluated by theamount of a product per gram of total protein (U/g-protein). Theactivity value (relative activity) of each mutant is presented in Table9, where a ratio of 100% corresponds to the 5-hydroxy-L-pipecolicacid-producing activity of the wild-type enzyme (McPH) without anymutation.

TABLE 9 Mutated Relative Mutant position activity (Wild type) — 100McPHm1 H4Q 82 McPHm2 F5Y 132 McPHm3 C23A 123 McPHm4 C44A 0 McPHm5 L99R18 McPHm6 V100A 2 McPHm7 D116G 27 McPHm8 C137A 42 McPHm9 D282E 152

Furthermore, the combination effect of the respective mutations wasexamined in the mutations corresponding to McPHm2, McPHm3, and McPHm9,each of which was effective to improve the activity. A plasmidexpressing a double mutant (McPHm10) with substitutions of phenylalanineto tyrosine at position 5 and cysteine to alanine at position 23, and aplasmid expressing a triple mutant, which is derived from the doublemutant and further has a substitution of aspartic acid to glutamic acidat position 282, were constructed. Then, recombinant E. coli strainswere produced according to the method described above, and the resultingenzyme solutions were used to evaluate the 5-hydroxy-L-pipecolicacid-producing activity. The result is presented in Table 10. The triplemutant McPHm11 (SEQ ID NO: 20; a gene encoding the same amino acidsequence is represented by SEQ ID NO: 19) exhibited an activity three ormore times higher than that of the wild-type enzyme.

TABLE 10 Mutated position Relative Mutant 1 2 3 activity (Wild type) — —— 100 McPHm10 F5Y C23A — 133 McPHm11 F5Y C23A D282E 314

As for McPHm10 and McPHm11, the hydroxylation activity was determined inaccordance with the method described in Example 4, where L-proline wasused as a substrate. Thus, virtually no activity against proline wasconfirmed in both cases.

1. A method of producing a hydroxy-L-pipecolic acid, the methodcomprising: allowing an L-pipecolic acid hydroxylase, a microorganism orcell having the ability to produce the enzyme, a processed product ofthe microorganism or cell, and/or a culture liquid comprising the enzymeand obtained by culturing the microorganism or cell, to act onL-pipecolic acid as a substrate in the presence of 2-oxoglutaric acidand ferrous ion, wherein the L-pipecolic acid hydroxylase has theproperties (1) and (2) below: (1) the enzyme can act on L-pipecolic acidin the presence of 2-oxoglutaric acid and ferrous ion to add a hydroxygroup to the carbon atom at positions 3, 4, and/or 5 of L-pipecolicacid; and (2) the enzyme has a catalytic efficiency (kcat/Km) withL-proline that is equal to or less than 7 times the catalytic efficiency(kcat/Km) with L-pipecolic acid.
 2. The method of producing ahydroxy-L-pipecolic acid according to claim 1, further comprising:allowing L-lysine and/or DL-lysine to react with (i-1) one or moreenzymes selected from the group consisting of an L-amino acid oxidase,an L-amino acid dehydrogenase and an L-amino acid aminotransferase, or(i-2) an amino acid racemase and one or more enzymes selected from thegroup consisting of a D-amino acid oxidase, a D-amino acid dehydrogenaseand a D-amino acid aminotransferase, for the production of3,4,5,6-tetrahydropyridine-2-carboxylic acid; and subsequently allowingan N-methyl-L-amino acid dehydrogenase to act on the3,4,5,6-tetrahydropyridine-2-carboxylic acid for the production of theL-pipecolic acid as the substrate.
 3. The method of producing ahydroxy-L-pipecolic acid according to claim 1, further comprising:allowing L-lysine to react with one or more enzymes selected from thegroup consisting of an L-lysine 6-oxidase, an L-lysine 6-dehydrogenaseand an L-lysine 6-aminotransferase, for the production of2,3,4,5-tetrahydropyridine-2-carboxylic acid; and subsequently allowinga pyrroline-5-carboxylate reductase to act on the2,3,4,5-tetrahydropyridine-2-carboxylic acid for the production of theL-pipecolic acid as the substrate.
 4. The method of producing ahydroxy-L-pipecolic acid according to claim 1, further comprisingallowing a lysine cyclodeaminase to act on L-lysine for the productionof the L-pipecolic acid as the substrate.
 5. The method of producing ahydroxy-L-pipecolic acid according to claim 1, wherein the content ofL-proline in the L-pipecolic acid as the substrate is not more than 10%(w/w).
 6. The method of producing a hydroxy-L-pipecolic acid accordingto claim 1, wherein the L-pipecolic acid hydroxylase further has theproperty (3) below: (3) the microorganism or cell having the ability toproduce the L-pipecolic acid hydroxylase, or the processed product ofthe microorganism or cell has a hydroxy-L-proline-producing activitythat is not more than 55%, where a ratio of 100% corresponds to thehydroxy-L-pipecolic acid-producing activity of the same microorganism orcell, or of the same processed product.
 7. The method of producing ahydroxy-L-pipecolic acid according to claim 1, wherein the L-pipecolicacid hydroxylase comprises the protein (A), (B), or (C) below: (A) aprotein having an amino acid sequence represented by SEQ ID NO: 2, 12,14, 16, 18, or 20; (B) a protein having the same amino acid sequence asthe amino acid sequence represented by SEQ ID NO: 2, 12, 14, 16, 18, or20 except that one or several amino acids are deleted, substituted,inserted, and/or added, which protein has the aforementioned properties(1) and (2); (C) a protein having an amino acid sequence with anidentity of not less than 80% to the amino acid sequence represented bySEQ ID NO: 2, 12, 14, 16, 18, or 20, which protein has theaforementioned properties (1) and (2).
 8. The method of producing ahydroxy-L-pipecolic acid according to claim 1, wherein the microorganismor cell having the ability to produce the L-pipecolic acid hydroxylaseis a microorganism or cell transformed with DNA encoding the L-pipecolicacid hydroxylase.
 9. The method of producing a hydroxy-L-pipecolic acidaccording to claim 8, wherein the DNA encoding the L-pipecolic acidhydroxylase comprises the DNA (D), (E), or (F) below: (D) DNA having anucleotide sequence represented by SEQ ID NO: 1, 11, 13, 15, 17, or 19;(E) DNA comprising the same nucleotide sequence as the nucleotidesequence represented by SEQ ID NO: 1, 11, 13, 15, 17, or 19 except thatone or several bases are deleted, substituted, inserted, and/or added,which DNA encodes a protein having the aforementioned properties (1) and(2); (F) DNA comprising a nucleotide sequence which hybridizes with acomplementary strand of the nucleotide sequence represented by SEQ IDNO: 1, 11, 13, 15, 17, or 19 under stringent conditions, which DNAencodes a protein having the aforementioned properties (1) and (2).